Defective Decidualization During and After Severe Preeclampsia Reveals a Possible Maternal Contribution to the Etiology

Defective decidualization during and after severe preeclampsia reveals a possible maternal contribution to the etiology

Tamara Garrido-Gomeza,b,c,d, Francisco Dominguezb, Alicia Quiñonerob, Patricia Diaz-Gimenob, Mirhan Kapidzicc,d, Matthew Gormleyc,d, Katherine Onac,d, Pablo Padilla-Isertee, Michael McMasterf, Olga Genbacevc,d, Alfredo Peralese,g, Susan J. Fisherc,d,h,i,1,2, and Carlos Simóna,b,g,j,1,2

aFundación Igenomix, 46980 Valencia, Spain; bInstituto Universitario IVI, Instituto de Investigación Sanitaria Hospital Clinico de Valencia INCLIVA, 46010 Valencia, Spain; cCenter for Reproductive Sciences, University of California, San Francisco, CA 94143; dDepartment of Obstetrics, Gynecology, and Reproductive Sciences, University of California, San Francisco, CA 94143; eDepartment of Obstetrics and Gynecology, Hospital Universitario La Fe, 46026 Valencia, Spain; fDepartment of Cell and Tissue Biology, University of California, San Francisco, CA 94143; gDepartment of Obstetrics and Gynecology, School of Medicine, Valencia University, 46010 Valencia, Spain; hThe Eli & Edythe Broad Center for Regeneration Medicine and Stem Cell Research, University of California, San Francisco, CA 94143; iDepartment of Anatomy, University of California, San Francisco, CA 94143; and jDepartment of Obstetrics and Gynecology, School of Medicine, Stanford University, Palo Alto, CA 94305

Edited by R. Michael Roberts, University of Missouri-Columbia, Columbia, MO, and approved August 11, 2017 (received for review April 20, 2017)

In preeclampsia (PE), cytotrophoblast (CTB) invasion of the uterus in studying the CTB subpopulation that invades the uterine wall in and spiral arteries is often shallow. Thus, the placenta’s role has the context of this syndrome. Targeted analyses of particular mo- been a focus. In this study, we tested the hypothesis that decidual lecular families, such as the vascular-type adhesion molecules that defects are an important determinant of the placental phenotype. are up-regulated as the extravillous CTBs enter the uterine wall, We isolated human endometrial stromal cells from nonpregnant revealed focal defects in differentiation (8). These results were donors with a previous pregnancy that was complicated by severe confirmed and augmented by global transcriptional profiling of PE (sPE). Compared with control cells, they failed to decidualize in CTBs isolated from the placentas of affected pregnancies as they vitro as demonstrated by morphological criteria and the analysis of differentiated along the invasive pathway over a period of 48 h in stage-specific antigens (i.e., IGFBP1, PRL). These results were bol- culture (9). The surprising finding that the abnormal pattern of stered by global transcriptional profiling data that showed they gene expression autocorrected to control levels by the end of the were transcriptionally inert. Additionally, we used laser microdissec- culture period pointed to a potentially important role for paracrine tion to isolate the decidua from tissue sections of the maternal–fetal effectors. In this regard, the decidua, which supports placental interface in sPE. Global transcriptional profiling revealed defects in growth and function, is a prime candidate. gene expression. Also, decidual cells from patients with sPE, which In humans, formation of the decidua does not depend on the dedifferentiated in vitro, failed to redecidualize in culture. Condi- presence of a conceptus (10). This progressive process, which tioned medium from these cells failed to support CTB invasion. To mimic involves hormonally regulated differentiation of human endo- aspects of the uterine environment in normal pregnancy, we added metrial stromal cells (hESCs), begins during the midsecretory PRL and IGFBP1, which enhanced invasion. These data suggested that phase of the menstrual cycle (11). The transformation is initiated failed decidualization is an important contributor to down-regulated in areas that are immediately adjacent to the uterine spiral ar- CTB invasion in sPE. Future studies will be aimed at determining teries, ultimately spreading throughout the entire endometrium whether this discovery has translational potential with regard to (12). In vivo and in vitro, this process is driven by increasing levels – assessing a woman’s risk of developing this pregnancy complication. of progesterone and local cAMP production (13 15), which

preeclampsia | human endometrial stromal cells | decidua | Significance cytotrophoblast | transcriptomics We provide evidence of a decidualization defect in the endo- reeclampsia (PE), which affects ∼8% of first-time pregnan- metrium of women with severe preeclampsia (PE) that was Pcies, impacts 8 million mother–infant pairs worldwide each detected at the time of delivery and persisted years after the year (1, 2). This complication, which is specific to human preg- affected pregnancy. We went on to link this defect to impaired nancy, is characterized by the new onset of hypertension, pro- cytotrophoblast invasion. The transcriptional signature of the teinuria, and other signs of maternal vascular damage such as defect could enable its detection before (or after) conception, edema (3). Severe PE (sPE) is diagnosed based on a further ele- which would aid the development of therapies focused on vation of blood pressure (systolic pressure ≥160 mm Hg or diastolic improving decidualization and perhaps preventing severe PE. pressure ≥110 mm Hg) or any of the following: thrombocytopenia, impaired liver function, progressive renal insufficiency, pulmonary Author contributions: O.G., A.P., S.J.F., and C.S. designed research; T.G.-G., F.D., A.Q., M.K., K.O., P.P.-I., O.G., and A.P. performed research; P.D.-G. and M.G. contributed new edema, and the new onset of cerebral or visual disturbances (4). reagents/analytic tools; T.G.-G., M.G., S.J.F., and C.S. analyzed data; P.P.-I. collected samples; Currently, the only definitive cure is delivery of the placenta and and T.G.-G., M.M., S.J.F., and C.S. wrote the paper. therefore the infant. As a result, PE accounts for 15% of preterm The authors declare no conflict of interest. births in the United States. Despite decades of research, a full This article is a PNAS Direct Submission. understanding of PE pathogenesis remains elusive, which com- Freely available online through the PNAS open access option. pounds the difficulties involved in the identification of predictive Data deposition: The data reported in this paper have been deposited in the Gene Ex- biomarkers and the development of targeted therapeutic strategies. pression Omnibus (GEO) database, https://www.ncbi.nlm.nih.gov/geo (accession nos. It is widely believed that the placenta plays a central role, with GSM2420587–GSM2480236). deficient cytotrophoblast (CTB) invasion of uterine spiral arte- 1S.J.F. and C.S. contributed equally to this work. rioles being a casual factor (5, 6). Currently, the pathogenesis of 2To whom correspondence may be addressed. Email: [email protected] or PE is conceptualized in a two-stage model, with the placental [email protected]. defect precipitating an abnormal maternal response that manifests This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. as the signs (7). Accordingly, there has been a great deal of interest 1073/pnas.1706546114/-/DCSupplemental.

E8468–E8477 | PNAS | Published online September 18, 2017 www.pnas.org/cgi/doi/10.1073/pnas.1706546114 Downloaded by guest on October 1, 2021 stimulate the synthesis of a complex network of intracellular and finding suggested that in vitro decidualization was impaired in PNAS PLUS secreted proteins that regulate decidualization (16). hESCs obtained from patients with former sPE compared with Morphologically, this process is characterized by the transforma- controls. tion of elongated fibroblast-like cells into an enlarged polygonal- or round-shaped population, a process that involves complex cyto- Alterations in the Global Transcriptional Profiles of Decidualized skeletal rearrangements (17). Actin, which is concentrated in the hESCs from Patients with Former sPE. Next, we used a microarray cortex of decidual cells, most often in a filamentous form (i.e., strategy to identify the molecular changes underlying the functional F-actin), regulates the intracellular reorganization and resulting decidualization defect found in hESCs from women who had ex- shape changes (18–20). Decidualization can also be viewed as a perienced sPE. Specifically, we carried out a transcriptomic analysis process whereby the cells acquire a secretory phenotype. Decid- of hESCs that were established from the normal pregnancy and sPE ualized hESCs secrete specific products such as prolactin (PRL) groups described earlier, nondecidualized and decidualized in vitro and insulin-like growth factor binding protein 1 (IGFBP1). Thus, (Fig. 2A). The clinical characteristics of the endometrial donors these proteins have been widely used as markers of decidualized are shown in SI Appendix,TableS2. An overview of the results is cells (21). They also play an important role in endometrial differ- presentedinFig.2B. In the nondecidualized state, only five statis- entiation and control of CTB invasion (22, 23). Overall deciduali- tically significant genes were differentially expressed between the zation helps regulate embryo implantation, and subsequently, CTB control and the sPE samples, and the fold differences were modest interactions with the uterus, making this process an essential com- (Fig. 2C). Thus, in a basal state, the hESCs from patients with ponent of establishing the maternal–fetal interface during normal former sPE were very similar to those from control women. pregnancy (24). Conversely, suboptimal decidualization can lead to During decidualization of the samples from control donors, aberrations in placentation and adverse pregnancy outcomes (25). the expression of 74 genes was significantly regulated based on a The uterine contribution to the etiology of sPE has received false discovery rate (FDR) <0.05 and at least twofold differentially little attention compared with the placenta’s role. Only recently, expressed (Fig. 2D; complete list provided in SI Appendix,TableS3). global transcriptional profiling of chorionic villus samples pointed They included genes known to be involved in decidualization. to insufficient or defective decidualization in pregnancies that were Some were up-regulated [e.g., CANNABINOID RECEPTOR 1 later complicated by sPE (26). In the present study, we hypothe- (CNR1), INSULIN RECEPTOR SUBSTRATE 2 (IRS2), and sized that aberrant decidualization could be an important con- MONOAMINE OXIDASE A (MAOA)] and others were down- tributor to the phenotypic alterations in placentation that are regulated [e.g., COCHLIN (COCH), LIM DOMAIN 2 (LMO2), associated with this pregnancy complication. To test this theory, we and ACTIN FILAMENT ASSOCIATED PROTEIN 1 (AFAP1L2)] used two approaches. First, we isolated hESCs from women with (16, 27–31). At the pathway level, processes that are relevant to previous sPE pregnancies and decidualized the cells in vitro. In decidualization, including regulation of oxygen responses, insulin parallel, we used laser microdissection to isolate decidual cells secretion, and proliferation, were up-regulated (SI Appendix, Fig. from the maternal–fetal interface of affected pregnancies. In S1A). No pathways were significantly down-regulated. both cases, global transcriptional profiling revealed profound Consistent with the results shown in Fig. 1, the comparison sPE-associated decidualization defects compared with controls. between nondecidualized and decidualized hESCs isolated from Furthermore, conditioned medium (CM) from freshly isolated patients with former sPE failed to detect the modulated expression decidual cells of women who experienced sPE failed to support of any genes [i.e., zero differentially expressed genes (DEGs); Fig. CTB invasion in vitro. To mimic the decidual microenvironment 2B]. In contrast, comparing the transcriptomes of the decidualized

during normal pregnancy, we added two secreted biomarkers of cells in the two groups revealed 129 misexpressed genes (at least MEDICAL SCIENCES decidual cells (PRL and IGFBP1), which improved CTB invasion. twofold; Fig. 2E; complete list in SI Appendix,TableS4). mRNAs Together, the results suggested that a hESC decidualization defect, with expression patterns that were validated by quantitative RT- which can be detected at the time of delivery for sPE, is evident PCR (qRT-PCR) analyses (SI Appendix,Fig.S2)aredenotedwith months to years after the affected pregnancy. Whether detection is asterisks in Fig. 2E and SI Appendix, Table S4.TheDEGincluded possible before the conception cycle of an sPE pregnancy remains to the up-regulation of mRNAs encoding molecules that are involved be determined. We propose that the gene signature that is associ- in hormone conversion (HSD17B2), extracellular structure orga- ated with defective decidualization in sPE could be used to explore nization (LAMA5, SULF1,andITGA11), vascular development this question in a high-risk population. Additionally, our findings (ANGPT2, EGR1,andRELAXIN2), and response to peptides suggested that therapies designed to improve decidualization could (KLF2, SSTR1,andIGBFP5; SI Appendix,Fig.S1B). The down- be a novel strategy in preventing this pregnancy complication. regulated category included genes that play important roles in decidualization (e.g., IGFBP1, CNR1,andIL-1B). The latter group Results functioned in numerous pathways such as cytokine–receptor inter- Failure of hESCs from Women with a Previous sPE Pregnancy to actions; response to wounding, inflammation, and estrogen (late); Decidualize in Vitro. We assessed decidualization of hESCs iso- and TFG-β signaling (SI Appendix,Fig.S1B). lated from endometrial biopsies of patients in whom sPE de- Finally, we analyzed the overlap between the DEGs in the veloped in a previous pregnancy (n = 13) compared with control nondecidualized vs. decidualized hESC comparison from women patients who had normal obstetric outcomes (n = 13). The ma- who had normal pregnancies (Fig. 2D) and the sPE vs. normal ternal and neonatal characteristics of the participants are sum- pregnancy group following decidualization (Fig. 2E). Fifteen marized in SI Appendix, Table S1. hESCs were decidualized by genes were up-regulated during normal hESC decidualization and treatment with cAMP and medroxyprogesterone acetate (MPA) down-regulated during decidualization of hESC from women with for 5 d. As experimental controls, cells from the same donor sPE. They included signaling molecules such as CANNABINOID were cultured in parallel without these additives. Localization of RECEPTOR 1 (CNR1), INSULIN RECEPTOR SUBSTRATE 2 F-actin in decidualized cells from women with uncomplicated (IRS2), and LYSOPHOSPHATIDIC ACID RECEPTOR 1 pregnancies showed the expected cytoskeletal reorganization (LPAR1); an ACTIN BINDING LIM PROTEIN FAMILY, and shape changes that were consistent with transformation from MEMBER 2 (ABLIM2); and A LATENT TRANSFORMING a fibroblast to a decidual phenotype (Fig. 1A). In contrast, hESCs GROWTH FACTOR BETA BINDING PROTEIN 1 (LTBP1) from women who had sPE failed to undergo these changes (Fig. with important functions during decidualization (SI Appendix,Fig. 1B). In nondecidualized hESCs, PRL (Fig. 1 C–E)andIGFBP1 S1C). In contrast, seven genes were down-regulated during normal (Fig. 1 F–H) levels detected in CM were low and not statistically decidualization and up-regulated in the equivalent samples from different between the two groups. As expected, secretion of both patients with former sPE (SI Appendix,Fig.S1D). They included molecules greatly increased upon decidualization of most of the molecules such as COAGULATION FACTOR C HOMOLOG, control cultures, but hESCs from patients with former sPE failed COCHLIN (COCH), and CORNICHON FAMILY AMPA to show this dramatic increase (Fig. 1 D, E, G, and H). This RECEPTOR AUXILIARY PROTEIN 3 (CNIH3). Thus, we concluded

Garrido-Gomez et al. PNAS | Published online September 18, 2017 | E8469 Downloaded by guest on October 1, 2021 Fig. 1. In vitro decidualization of hESCs was impaired in patients with previous pregnancies that were complicated by sPE. hESCs were obtained from nonpregnant women, patients with former sPE, or subjects who had a normal (norm) pregnancy outcome. The cells were decidualized by culturing for 5 d in the presence of cAMP and MPA. Nondecidualized cultures were maintained in the same medium without additives. (A) Localization of F-actin by rhodamine- phalloidin staining of hESCs from women with uncomplicated pregnancies showed the expected cytoskeletal reorganization and shape changes that were consistent with transformation from a fibroblast to a decidual phenotype. (B) hESCs from women who had sPE failed to undergo these changes. (C and D) PRL and (F and G) IGFBP1 secretion. (E and H) Summary of the ELISA data. Low levels of both molecules were detected in the CM of hESCs from the two donor groups before decidualization (solid bars). PRL and IGFBP1 levels greatly increased in most cultures of decidualized hESCs from donors who had normal pregnancy outcomes (gray hashed bars). In contrast, hESCs from patients with former sPE failed to show the decidualization-related increases in secretion of these molecules (black hashed bars; **P < 0.01 and ***P < 0.005; n.s., nonsignificant). (Scale bar: 100 μm.)

that the genes that had discordant expression patterns in decid- IN THE BLOOD), OGN (PROTEOGLYCAN), and COL8A1 ualized hESCs from the two groups could play important roles in the (COLLAGEN VIII; major component of membrane corneal failed in vitro transformation of cells from patients with former sPE. endothelium). Given the functions of defensins, we surmised that these genes were up-regulated in nPTB rather than down-regulated Molecular Defects in Situ of Decidua Basalis or Parietalis from Control in sPE. The up-regulation of a select natural antimicrobial agent in vs. sPE Pregnancies. We used a laser-microdissection approach to the nPTB samples is likely attributable to the start of an infectious isolate portions of the decidua basalis (DB) or decidua parietalis process that has not yet manifested in terms of the clinical signs (32, (DP). Cells were captured from tissue sections of biopsy speci- 33). The low yield of DEGs in this dataset was likely the resul ot the mens from cases of women with sPE vs. control women [i.e., numerous cell types that comprise the DB and the challenges of gestational age-matched samples from women who had a capturing decidual cells from this complex mixture. No pathways spontaneous preterm birth with no signs of infection (nPTB); were significantly up- or down-regulated. Fig. 3A]. The clinical characteristics of the participants are In contrast, the clear boundary between the smooth chorion summarized in SI Appendix,TableS5. An overview of the results is and the DP enabled efficient laser microdissection of the latter shown in Fig. 3B. In the DB, 79 genes were significantly differen- cells. Comparison of heat maps of the mRNA samples that were tiallyexpressedinsPEvs.nPTBwithmodestfoldchanges(Fig.3C; isolated from sPE cases vs. nPTB controls revealed 227 genes full list in SI Appendix,TableS6). They included the up-regulation that were differentially expressed in sPE by at least twofold (Fig. of mRNAs encoding molecules involved in RNA processing. Down- 3D; complete list in SI Appendix, Table S7). The up-regulated regulated genes included DEFB1 (MICROBICIDAL AND genes encoded molecules with immune functions such as PRG2 CYTOTOXIC PEPTIDE), CP (COPPER-CARRYING PROTEIN and KLRF1. Other genes in this category included RNASE2,

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Fig. 2. Global transcriptional profiling confirmed failed decidualization of hESCs obtained from nonpregnant women with a previous pregnancy complicated by sPE. (A) Schematic drawing of the study design. hESCs were isolated from endometrial biopsies and decidualized in vitro. The donors were nonpregnant women with previous normal pregnancy outcomes or patients with former sPE. (B) Summary of the LIMMA paired comparisons showing the number of DEGs by FDR <0.05 and at least twofold between the groups. (C) Before decidualization, there were five DEGs between hESCs from the normal pregnancy outcome group vs. patients with former sPE. (D) Heat map showing the 50 most highly DEGs (total = 74; SI Appendix, Table S3) that were modulated during decidualization of hESCs from donors who had normal pregnancy outcomes. (E) Heat map showing the 50 most highly DEGs (total = 129; SI Appendix, Table S4) that were misexpressed following decidualization of hESCs from donors with a former sPE pregnancy compared with those with normal pregnancies (*mRNA expression patterns validated by qRT-PCR; Δ, fold change).

PZP, PDGFD, a Wnt inhibitor (NOTUM), and PROM1, which with the adjacent DP. In all cases, the protein-level results confirmed plays a role in the maintenance of adult stem cells. AOX1, which the expression patterns that were suggested by the transcriptomic data catalyzes the formation of superoxide and NO, was also up-regulated, (SI Appendix,Fig.S4). a possible sign of oxidative stress. At a pathway level, regulation of cell communication, several metabolic processes, and transmembrane Absence of Decidualization Markers in sPE Pregnancies. To analyze receptor protein tyrosine kinase signaling (among others) were sig- decidualization in situ, we assessed PRL (Fig. 4 A and B) and nificantly up-regulated (SI Appendix,Fig.S3). IGFBP1 (Fig. 4 C and D) expression in tissue sections of the DB The down-regulated mRNAs included ILs (CXCL8, IL23A, and the DP in sPE (n = 5) compared with nPTB (n = 4). The IL1A), CXCL5, as well as proteinases and their inhibitors (SPINK1, clinical characteristics of the pregnancies are summarized in SI ADAMTS4,andMMP10) that play important roles during decidu- Appendix, Table S8. CTB identity was confirmed by anti- alization. At a pathway level, regulation of cell adhesion, locomo- cytokeratin 7 (CK7; Fig. 4 A–F) immunoreactivity, and stro- tion/migration, morphogenesis, extracellular structure, and immune mal cells were visualized with anti-vimentin (VIM; Fig. 4 E processes were impacted (SI Appendix,Fig.S3). However, neither and F). The results showed that PRL and IGFBP1 were broadly PRL nor IGFBP1 were identified as misexpressed at the mRNA expressed by decidualized stromal cells (DB and DP) in control level. This suggested that a posttranslational mechanism could be nPTB samples (Fig. 4 A and C). In contrast, expression of both involved in repressing their expression at the protein level (34). decidualization markers was greatly reduced and, in many in- We validated the microarray results at the protein level for stances, absent in the sPE samples (Fig. 4 B and D). These data three DEG (PEG1/MEST and PRG2,up-regulatedinsPE;BMP2, were additional evidence that sPE is associated with widespread down-regulated in sPE). In these experiments, we used an immuno- defects in decidualization that were evident in samples obtained localization approach applied to tissue sections of the fetal membranes immediately after delivery.

Garrido-Gomez et al. PNAS | Published online September 18, 2017 | E8471 Downloaded by guest on October 1, 2021 Fig. 3. Global transcriptional profiling of the DB and the DP revealed the DEGs in sPE vs. control pregnancies. (A) Schematic drawing of the study design. Laser microdissection enabled isolation of portions of the DB from the basal plate and DP, which was adjacent to the fetal membranes. (B) Summary of the LIMMA paired comparisons showing the number of DEGs between equivalent decidual compartments in sPE vs. nPTB (FDR < 0.05 and fold change ≥ 2). (C) Heat map showing the 50 most highly DEGs (total = 79; SI Appendix, Table S6) in the DB of nPTB vs. sPE patients. (D) Heat map showing the 50 most highly DEGs (total = 227; SI Appendix, Table S7) in the DP of nPTB vs. sPE patients.

Failure of Decidualization Marker Expression in Freshly Isolated These results constituted additional evidence of defective decidu- Stromal Cells from sPE Decidual Biopsies. Next, we isolated decidual alization in sPE. cells from sPE (n = 5) or nPTB cases (n = 4) with the goal of determining their status in terms of expressing stage-specific antigens Failure of Stromal Cells Isolated from Decidual Biopsy Specimens that are typically associated with these cells. Freshly isolated stromal Obtained at Delivery from Patients with sPE to Redecidualize in cells that were cultured overnight did not react with antibodies that Vitro. Next, we asked whether the isolated stromal cells that were were specific for markers of endothelial or hematopoietic cells, cultured for three to five passages could redecidualize in vitro. To including macrophages. Immediately after plating, rhodamine- answer this question, we monitored morphological changes and phalloidin immunostaining showed the expected pattern of F-actin secreted biomarkers (PRL and IGFBP1) of this process after 5 d of distribution in polygonal/round cells that were isolated from the DB hormone treatment. In cells from nPTB control patients, regardless or DP from control nPTB samples (Fig. 5A). In contrast, stromal of their compartment of origin, decidualization was associated with cells from decidual biopsy specimens of patients with sPE had an a characteristic polygonal/round phenotype as demonstrated by elongated morphology with a fibroblast-like F-actin organization rhodamine-phalloidin immunostaining (Fig. 6A). In contrast, (Fig. 5B). Immunostaining with anti-PRL (Fig. 5 C and D)oranti- stromal cells from patients with sPE failed to display morpho- IGFBP1 (Fig. 5 E and F) showed that VIM-positive stromal cells logical changes during redecidualization (Fig. 6B). In control from sPE deciduas (Fig. 5 G and H) had much lower antibody cells following decidualization, secretion of PRL (Fig. 6C) and reactivity than was observed in the control nPTB samples. Finally, IGFBP1 increased (Fig. 6D). In contrast, the equivalent cells we quantified the cell secretion of PRL (Fig. 5I) and IGFBP1 (Fig. isolated and cultured from patients with sPE failed to increase 5J) after overnight culture. In nPTB, production of both molecules secretion of either molecule (Fig. 6 C and D) in response to was higher by cells isolated from the DP compared with the DB. In MPA and cAMP treatment. Thus, isolation and culture did not comparison, sPE was associated with a dramatic reduction in PRL rescue defective decidualization of stromal cells from patients and IGFBP1 secretion by cells isolated from both compartments. with sPE.

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Fig. 4. sPE is associated with a striking down-regulation of PRL and IGFBP1 expression in the decidua (DEC). Tissue sections of the maternal/fetal interface that contained portions of the DB or the DP were coimmunostained with an antibody against CK7, which enabled visualization of CTBs, and decidual markers PRL (A and B) and IGFBP1 (C and D). (E and F) Adjacent sections were stained with anti-VIM, which labeled DEC cells. Nuclei were visualized with DAPI. In nPTB samples, anti-PRL and anti-IGFBP1 gave strong signals in the DB and DP (CK7-negative, VIM-positive). In contrast, the VIM-positive DEC cells in sPE samples failed to react with anti-PRL and anti-IGFBP1 or stained weakly for these markers. (G and H) The immunolocalization results shown in A–D were semi- quantified in ImageJ. Representative areas (n = 3 per sample) of the DB and DP from nPTB and sPE cases were subjected to ImageJ analyses. The results (intensity/area [0.3 mm2]) were expressed relative to the data from the control/nPTB DB samples. Data are expressed as the mean ± SEM (*P ≤ 0.05, **P ≤ 0.01,

and ***P ≤ 0.001). Three or four representative areas of each sample were analyzed (sPE, n = 5 cases; nPTB, n = 4 cases). Am, amnion; iCTBs, invasive CTBs; MEDICAL SCIENCES schCTBs, smooth chorion CTBs. (Scale bars: 100 μm.)

Failure of CM from sPE Decidual Cells to Promote CTB Invasion. To shallow placentation that is frequentlyobservedinthispregnancy understand the functional impact of the sPE-associated decidu- complication. In vitro, CTB invasion could be significantly increased alization defect, we asked whether this phenomenon was mech- by adding two secreted proteins that are highly expressed in normal anistically related to reduced CTB invasion in this pregnancy decidua and significantly reduced in sPE, evidence of their effects on complication. As our approach, we isolated stromal cells from placental cells. samples of the DB and the DP of sPE (n = 4) or control nPTB (n = 3) cases. They were cultured overnight. Then, the CM was Discussion collected, and an sPE-associated down-regulation of PRL and Together, the data presented here suggested that hESCs from IGFBP1 secretion in sPE compared with nPTB cultures was patients with sPE, in the nonpregnant state and at the end of confirmed (SI Appendix, Fig. S5). Accordingly, experimental or pregnancy, failed to decidualize. Initial experiments used an in control CM was added to second-trimester CTBs (n = 10 pla- vitro approach. Before decidualization, hESCs isolated from centas), which were cultured on a Matrigel substrate. Invasion was patients in whom sPE developed, or from control women with a assayed by counting the number of CTBs or their cellular processes previous normal pregnancy, had similar morphological features that reached the underside of the filters (Fig. 7A). In the presence and patterns of PRL and IGFBP1 secretion. In keeping with these of the control (i.e., nPTB) CM, robust invasion was observed, observations, they had nearly identical transcriptomes. However, which was not statistically different between the CTBs that were they differed in their ability to decidualize in culture. By mor- cultured in the DB or the DP samples (Fig. 7B). In contrast, the phological criteria and marker expression (PRL and IGFBP1), the CM from the equivalent stromal-cell populations of sPE cases did cells in the group that formerly experienced sPE resisted decidu- not support CTB invasion. alization, which was confirmed by microarray analyses that showed Finally, we sought to establish a link between reduced PRL that they were transcriptionally inert. Global transcriptional profiling and IGFBP1 secretion by decidualized stromal cells in the setting identified 129 genes as differentially expressed. Approximately 20% of sPE and the inability of CM from these cells to stimulate CTB (22 of 129) were inversely regulated, suggesting that they might play invasion. When the cells were cultured in fresh medium rather particularly important roles in normal decidualization. The mis- than CM, few CTBs reached the filter undersides (Fig. 7C). This regulated genes functioned in many biological processes, including suggested that the nPTB decidual cells released factors that extracellular structure organization, tissue development, and respon- promoted this process. The addition of PRL or IGFBP1 (10 ng/mL) ses to wounding, oxygen, and inflammation. to fresh medium had little effect. However, acting together, these This finding prompted us to ask whether there was evidence of two factors significantly increased invasion. Thus, the intrinsic de- defective decidualization at the end of pregnancies that were cidual stromal defects that were observed in sPE had paracrine ef- complicated by sPE, which necessitated delivery in the preterm fects on CTBs that were consistent with the reduced invasion and period. As gestation-matched controls, we analyzed equivalent

Garrido-Gomez et al. PNAS | Published online September 18, 2017 | E8473 Downloaded by guest on October 1, 2021 Fig. 5. Freshly isolated stromal cells from decidual biopsy specimens of patients with sPE displayed decidualization defects in culture. Cells were isolated from the DB or DP and analyzed at p0. Donors were women whose pregnancies were complicated by nPTB (n = 4) or sPE (n = 5). (A and B) The F-actin cytoskeleton of the cells was visualized by rhodamine-phalloidin staining, and nuclei were imaged with DAPI. In nPTB pregnancies, cells from either decidual compartment had a polygonal shape with a complex, well-developed network of actin filaments. (B) In contrast, the cells from sPE pregnancies were flattened with a much less well-developed actin cytoskeleton. Immunolocalization of PRL (C and D), IGFBP1 (E and F), and VIM (G and H). (C, E, and G) VIM-positive cells from the nPTB donors gave strong signals for the decidualization markers that were evaluated. (D, F,andH) In contrast, few of the VIM-positive cells from patients with sPE reacted with anti-PRL or anti-IGFBP1. (I and J) Secretion of PRL and IGFBP1 mirrored the immunostaining results. Data are the mean ± SEM of each sample, which was analyzed in triplicate (*P < 0.05, **P < 0.01, and ***P < 0.001). (Scale bars: 100 μm.)

samples from women who had an nPTB, a study design that has pregnancy, making this the period in which dysregulated decidual enabled us to pinpoint changes in gene expression that are gene expression could play a role in sPE-associated defects in these specific to sPE (9, 35). Our goal was to use laser microdissection processes. In this regard, a previous study analyzed gene expression to isolate cells from both areas of the decidua. Attempts to obtain by chorionic villous samples that were obtained near the end of the a pure population from the DB, which contains a mixture of ma- first trimester from pregnancies in which the women went on to ternal and fetal cells, was relatively unsuccessful, as shown by the develop sPE (26). The comparator was equivalent samples from fact that a substantial number of the differentially expressed genes women who had normal obstetric outcomes. The genes that were encoded known CTB proteins. In contrast, as a result of the clear dysregulated in the sPE group were decidual rather than tropho- demarcation between the CTB layer of the smooth chorion and the blast in origin, overlapping with those that are modulated during DP, the laser-capture approach was successful in isolating the normal decidualization in mid/late-secretory endometrium from a latter cells. In all, 227 genes were differentially expressed in sPE vs. nonconception cycle and endometrium from tubal ectopic preg- nPTB. These results suggested a striking sPE-associated dysregu- nancies. The majority (73%) changed in the opposite direction of lation of gene expression in the DP. In accordance with this finding, their regulation during normal endometrial maturation. They included immunoanalyses showed that cells of the DB and DP failed to IGFBP1 and PRL, which we also showed were down-regulated at the express PRL and IGFBP1. Following isolation, passage in culture, protein level. Analysis of the overlap with the dysregulated genes and subsequent dedifferentiation, theyalsofailedtoredecidualize. identified in the present study (in vitro and in situ) identified nine Finally, we wondered whether the sPE-associated decidual defect commonalities. They included EGR1, IGFBP1, IL15, IL1B,and could impact CTB invasion, as is frequently observed in the basal NTN4 (NETRIN). plates of sPE pregnancies. In these experiments, CM from cultured Additionally, Rabaglino et al. reported evidence of natural cells established from the DP or DB following delivery as a result of killer (NK) cell dysfunction (26). Likewise, we found differential sPE markedly reduced CTB invasion compared with the equivalent expression of immune cell genes in microdissected decidual nPTB samples (Fig. 7B). The combined addition of PRL and IGFBP1 samples from patients with sPE, evidence that maternal leuko- increased CTB invasion (Fig. 7C). Thus, we linked a potential cause of cytes are found within the uterine lining. NK cell receptors sPE—failed decidualization and a suboptimal uterine microenviron- (KLRF1 and KIR2DL2) and T-cell receptors (TRDJ3, TRAJ5, ment—to a potential downstream effect: inhibition of CTB invasion. and TRAJ59) were misexpressed in our datasets. Thus, concor- Nevertheless, it is important to note that CTB invasion and dance between the data presented by Rabaglino et al. (26) and in remodeling of the spiral arteries largely occurs in the first half of the present paper bolstered the conclusion that sPE is associated

E8474 | www.pnas.org/cgi/doi/10.1073/pnas.1706546114 Garrido-Gomez et al. Downloaded by guest on October 1, 2021 PNAS PLUS

Fig. 6. Cultured hESCs from decidual biopsies of patients with sPE failed to redecidualize in vitro. Cells were isolated from the DB or DP and analyzed at p3–p5, i.e., after they lost expression of the decidualization markers PRL and IGFBP1. Donors were women whose pregnancies were complicated by nPTB (n = 3) or sPE (n = 4). Redecidualization was induced by treatment with cAMP (0.5 μM) and MPA (1 μM). CM and cells were analyzed after 5 d. (A and B) Visualization of the F-actin

cytoskeleton via rhodamine-phalloidin staining. Nuclei were imaged with DAPI. When hESCs from donors who gave birth with nPTB were redecidualized, they MEDICAL SCIENCES enlarged, becoming polygonal/round with a complex actin cytoskeleton. In contrast, hESCs from donors with sPE failed to undergo the expected shape changes, often exhibiting disorganized actin cytoskeletons. (C and D) PRL and IGFBP1 secretion, measured by ELISA, were consistent with failed decidualization of hESCs from patients with sPE. Data are the mean ± SEM of each sample, which was analyzed in triplicate (*P < 0.05 and **P < 0.01; n.s., not significant). (Scale bar: 100 μm.)

with a defect in decidualization, which is evident early in pregnancy increased risk of developing this syndrome, whereas a paternal when CTBs are remodeling the spiral arteries, and at birth. Also, history has no effect (39). Twin studies suggest the role of ma- both studies found evidence of differential gene expression by ternal and fetal gene interactions (40). Whether this is evidence maternal immune cells that populate the decidua. of an inherited decidualization defect remains to be determined. Spontaneous endometrial decidualization followed by pro- Regarding the link between sPE in a first pregnancy and a sub- gesterone withdrawal, which “triggers” shedding, is a major feature sequent reduction in infertility, a definitive answer has been of the endometrium in menstruating species (e.g., humans, old- elusive because women who experience the severe form of this world primates, elephant shrews, and fruit bats). In contrast, the pregnancy complication are less likely to attempt another preg- endometrium of nonmenstruating mammals decidualizes only if nancy (41). However, the association of recurrent PE with a thereiscontactbetweenanembryoandtheuterus,i.e.,uponim- longer interpregnancy interval may be evidence of subfertility in plantation. Thus, in humans, the decidua controls conception and this group (42, 43). Interestingly, some patients with normal first- the course of pregnancy. In nonmenstruating species, the embryo time deliveries go on to develop sPE in subsequent pregnancies. controls this process as exemplified by delayed implantation in We speculate that the reasons could include an age-related decline mice (36). Considered in this context, our in vitro and in situ data in the ability of endometrial stromal fibroblasts to decidualize. This suggested that sPE is associated with a marked decidualization could be a primary cause or occur secondarily as the result of a defect that the embryo is sometimes able to overcome, thereby previous Cesarean section (44) or obesity (45). Finally, in women establishing pregnancy. In this scenario, the extravillous CTB sub- who experience sPE, subsequent normal pregnancies usually out- population penetrates the uterine wall and spiral arteries to the number cases of recurrent PE. Nevertheless, recurrence rates as high degree that is required to sustain pregnancy for varying periods of as 65% have been reported (46). We speculate that some women in time, but short of the duration of normal pregnancy. This may be a this group might regenerate a more normal endometrium during the result of the highly invasive nature of these placental cells (37), extensive remodeling of the uterine lining that takes place after which enables them to anchor not only to the uterus, but also to delivery. Alternatively, these may be pregnancies that are associated ectopic sites that they penetrate in search of a blood supply (38). with faulty placentation rather than abnormal decidualization. Thus, we concluded that failed decidualization restricts the depth of With regard to clinical practice, the beneficial effects of removing CTB invasion such that pregnancy is maintained but ends in sPE. the placenta in an sPE pregnancy are well known, but removing the Several other types of data support this theory. A decidual role decidua in the immediate postpartum period via uterine curettage in PE has been suggested by epidemiological studies. For ex- also aids in resolution of the signs, increasing the rate at which mean ample, a maternal history of PE is associated with a 24–163% arterial pressure decreases and platelet counts increase (47, 48).

Garrido-Gomez et al. PNAS | Published online September 18, 2017 | E8475 Downloaded by guest on October 1, 2021 Fig. 7. CM from decidual cells of patients with sPE inhibited CTB invasion in vitro. (A) Diagram of the experimental design. Decidual cells were isolated from the DB or DP and cultured overnight, and the CM was then isolated. The donors were women whose pregnancies were complicated by nPTB (n = 3) or by sPE (n = 4). CTBs were isolated from second-trimester placentas (15–17 wk, n = 4; 18–20 wk, n = 3; 21–23 wk, n = 3). They were cultured (72 h) on Matrigel-coated Transwell filters in medium conditioned by the nPTB or sPE decidual cells. CTBs and cellular processes that reached the undersides of the filters were counted. (B) Compared with the equivalent nPTB samples, CM from the cells of sPE donors significantly inhibited CTB invasion regardless of whether they were isolated from the DB or DP. (C) The addition of PRL and IGFBP1 (10 ng/mL each) to fresh medium increased CTB invasion to the levels that were observed when the cells were incubated in CM from nPTB cultures. Data are expressed as the mean ± SEM of duplicate wells (**P < 0.01 and ***P < 0.001; n.s., not significant).

Likewise, curettage exacerbates the decrease in circulating levels of cycle via an endometrial biopsy, thus enabling estimation of PE sFlt-1 (49), a biomarker that has been mechanistically linked to the risk in a subsequent pregnancy. In this regard, our results sug- signs of this syndrome (50–52). In this regard, overexpression of gested that IGFBP1 and PRL assays at the protein level could be sFlt-1 by decidual cells produces a PE-like syndrome in mice (53). useful. Other dysregulated mRNAs may also be biomarkers, Finally, a decidualization defect could help explain our previous suggesting the possibility of the development of a gene-expression finding that isolation and culture of CTBs from sPE placentas re- signature for the prospective diagnosis of a decidualization defect. versed the gene dysregulation that was initially observed (9). In light Such a test would open the door to possible therapeutic inter- of the data presented here, this may be evidence that a suboptimal ventions, which could involve pharmacological or other treatments uterine environment lies upstream of failed CTB differentiation and to restore a normal decidual phenotype before pregnancy. Given invasion. This conclusion is also supported by our finding that CM that failed decidualization is likely associated with a spectrum of from the faulty decidualized stroma in sPE restricted CTB invasion — B reproductive defects including failed implantation and mis- (Fig. 7 ), which could be stimulated by the addition of two factors — that decidual cells normally secrete (Fig. 7C). carriage it could be important to determine whether a subset of The findings presented here show a decidualization defect that patients with these conditions also benefit from the restoration of is evident at the time of delivery as a result of sPE and lingers for normal decidualization. Also, women with endometriosis who years afterward. Our work challenges the concept that sPE is exhibit decidualization resistance would be an interesting pop- primarily a disorder of first pregnancies and provides an expla- ulation to evaluate prospectively for possible treatment of infertility. nation of why women with sPE are more prone to a recurrence in Thus, the results of the work presented here suggest new directions subsequent pregnancies. Accordingly, the results of this study for research into the causes of PE and possible strategies for the could have interesting translational potential. It is possible that development of clinically useful tests that enable prospective risk failure to decidualize could be detected during a nonconception evaluation and possible treatment.

E8476 | www.pnas.org/cgi/doi/10.1073/pnas.1706546114 Garrido-Gomez et al. Downloaded by guest on October 1, 2021 Materials and Methods made this study possible; USCF recruiters Mss. Lisa Wilson, Lisa Gertridge, Allison PNAS PLUS O’Leary, Stephanie Leong, Jean Perry, and Rachel Freyre; Ms. Laura Rubert hESCs were isolated and cultured from endometrial biopsies. Their ability to (Hospital La Fe) for assistance in compiling the clinical data; the patient partic- decidualize in vitro was profiled by monitoring the expression of stage- ipants; and Ms. Norma McCormack and Mr. Harry Slomovits in preparing and specific antigens and by global transcriptional profiling. Laser microdissection submitting this paper. This work was supported by fellowship CD14/00229 from enabled the enrichment of decidual cells from the maternal-fetal interface. the Spanish Carlos III Institute, through the Sara Borrell Programme (to T.G.-G.); ’ The cells transcriptome was profiled immediately after isolation and fol- Spanish Generalitat Valenciana Grant GV/2016/033 (to T.G.-G.); Miguel lowing decidualization in vitro. The effect of soluble factors released from Servet Spanish Program Grant CP13/00075 cofounded by European Regional freshly isolated decidual cells on cytotrophoblast invasion was determined Development Funds (FEDER) (to F.D.); and European Regional Development by assaying the cells’ ability to penetrate Matrigel-coated Transwell filters. Funds (FEDER) and the Spanish Ministry of Economy and Competitiveness See SI Materials and Methods for detailed information. (MINECO), Grant SAF2015-67154-R (to C.S.). This work was also supported by 1 R37HD076253 (to S.J.F.) and by the Eunice Kennedy Shriver National Institute ACKNOWLEDGMENTS. We thank Dr. Mari-Paule Thiet and the UCSF Maternal- of Child Health & Human Development of the National Institutes of Health Fetal Medicine Division for invaluable help in obtaining the tissue samples that under Award P50HD055764 (to S.J.F.).

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